The ability of cells to migrate toward a target location plays an important role in many biological processes such as tissue and organ development, wound healing, and the tracking and capture of invaders by the immune system. Cell migration involves changes in shape of the cell, dynamic changes in the adhesion to the surrounding, and signaling cues that provides directional guidance. Chemical signaling cues are well known and are being studied in detail: Receptor binding of chemo attractants triggers intracellular signaling pathways that amplify the chemo attractant signal and trigger cell migration and other processes. Recent work has shown that mechanical stimuli also have a profound effect on cell behavior, including the demonstration that one may guide cell differentiation toward a target cell type by plating cells on surfaces with stiffness comparable to the target cell type. It is becoming increasingly clear that nanotopography also provides an important mechanical stimulus for cells. Surfaces with peaks and valleys of size tens of nm tend to enhance the activity of the actin cytoskeleton in many recent studies. However, little is known about how nanotopography affects cytoskeletal activity, and to what degree nanotopographic stimuli affect specific cytoskeletal functions, in particular cell migration. We propose to use the model organism Dictyostelium discoideum to understand, how nanotopographic cues affect cell migration at many levels, from the molecular level of intracellular signals, to the shapes and migration dynamics of whole cells, to the collective behavior of cell groups. The research proposed here will address the following hypotheses: Hypothesis 1: Nanoscale surface features (nanotopography) trigger biochemical signals that influence cell motility and affect chemotactic signaling pathways. Nanotopography also affects larger-scale phenotypes, in particular collective behavior of groups of cells. Hypothesis 2: Spatial patterning of the nanotopography of a surface can direct motile cells (as an alternative to, or in combination with, chemical signals). Hypothesis 3: Nanotopography affects cell migration along fibers and through three dimensional fiber networks. To test these hypotheses, our specific aims are: Aim 1: Measure and quantify the effects that nanopatterned, 2D surfaces have on intracellular signals, shape and motility of individual cells, and collective cell migration. Aim 2: Determine how spatial patterning of nanotopography directs cell migration. Aim 3: Analyze the influence of nanoscopic and microscopic geometry on cell motion in three dimensions, using 3D synthetic fiber networks with controlled nanotopography.